1. Technical Field
This invention relates to plasmid-borne genetic markers and related processes useful for introducing exogenous DNA to yeast strains, particularly Saccharomvces cerevisiae.
2. Description of the Prior Art
Selective modification of the genetic complement of organisms by recombinant DNA techniques has enabled efficient production of valuable proteins, introduction of new capabilities to recipient organisms, and optimization of existing characteristics of organisms. To date, these techniques have been primarily applied to bacteria. particularly Escherichia coli. However, eukaryotic organisms, especially yeasts such as Saccharomyces cerevisiae, can also be employed as recipients of recombinant DNA molecules. Certain strains of Saccharomvces, particularly those bred for use in the brewing and baking industries, are of considerable commercial importance. Optimization or modification of industrial yeast varieties by recombinant DNA techniques could significantly increase their value.
In a typical cloning experiment, a selected DNA segment is incorporated into an autonomously replicating DNA molecule known as a vector. In a process called transformation, a recipient cell population is contacted with a preparation of vector molecules under conditions permitting incorporation of vector molecules by recipient cells. If a vector molecule contains a "marker" gene capable of expression by the recipients, those cells which have actually incorporated a vector molecule can be identified. For example, if a vector contains a gene conferring resistance to a particular antibiotic, transformed cells will be identifiable by their ability to grow and multiply in the presence of the antibiotic. Alternatively. a vector molecule can comprise a gene complementing a mutation which has eliminated the capacity of the recipient strain to synthesize a particular nutrient (a condition known as auxotrophy). In this case, only those cells which have incorporated and expressed a vector-borne nutritional marker gene will grow and multiply in media lacking the nutrient.
Experiments involving transformation of wild-type or industrial yeast strains have been impeded by unavailability of suitable genetic markers for detection of transformants. Industrial yeast strains generally lack auxotrophic mutations, which would provide means for selecting transformed cells. Moreover, few suitable antibiotics or other growth-affecting compounds for which resistance phenotypes are available are now known. Several marker systems are summarized below.
Certain genes of bacterial origin have been successfully employed as selective markers in yeast. Reipen, et al., Current Genetics 6:189 (1982) disclose use of a gene encoding beta-lactamase, an antibiotic-inactivating enzyme derived from E. coli, as a selectable marker in transformation experiments involving Saccharomyces cerevisiae. This gene was effective, however, only when expressed under control of a promoter sequence derived from another yeast operon. Rose, et al., Proc. Nat. Acad. Sci. USA 78:2460 (1981), disclose fusion of an intact URA3 gene from S. cerevisiae and a beta-galactosidase gene from E. coli on a hybrid plasmid cloning vehicle. Yeast transformed with the hybrid plasmid expressed beta-galactosidase activity. Jiminez, et al., Nature 287:869 (1980) describe experiments in which S. cerevisiae was co-transformed with a yeast vector and a bacterial colicin derivative carrying a transposable element conferring resistance to 2-deoxystreptamine. a relatively potent inhibitor of protein synthesis. Cohen. et al., Proc. Nat. Acad. Sci. USA 77:1078 (1980) describe transformation of S. cerevisiae to chloramphenicol resistance With a chimeric plasmid bearing an E. coli chloramphenicol resistance determinant. Gritz, et al., Gene 25:179 (1983) disclose transformation of yeast with a hybrid plasmid containing an E. coli gene conferring selectable resistance to the antibiotic hygromycin-B.
Although hybrid plasmids comprising yeast and bacterial DNA fragments have been employed in the research setting, circumstances can be envisioned (e.g., production of new strains for food processing) in which it would be desirable to avoid introduction of bacterial DNA into a particular yeast strain. In such a case, a vector-borne selectable marker of yeast origin would be preferred. The following references disclose plasmids bearing dominant selectable markers of yeast origin.
Fried, et al., Proc. Nat. Acad. Sci. USA 78: 238 (1981) describe experiments in which S. cerevisiae resistant to a protein synthesis inhibitor, trichodermin, were produced by transformation of a sensitive host strain with a plasmid population containing a library of DNA fragments derived from a resistant yeast strain.
Another approach to construction of antibiotic- or inhibitor-resistant yeast phenotypes involves gene amplification, in which multiple copies of a gene encoding an enzyme targeted by a given inhibitor are introduced to the host cell. A resulting increase in enzyme activity can result in a selectable resistance phenotype. For example, Rine, et al., Proc. Nat. Acad. Sci. USA 80:6750 (1983) describe gene amplification experiments leading to S. cerevisiae transformants resistant to the growth inhibitors tunicamycin and compactin. Fogel, et al.. Proc. Nat. Acad. Sci. USA 79:5342 (1982) describe selection of copper-toxicity resistant S. cerevisiae from a recipient strain transformed with a library of random yeast genomic DNA fragments inserted in an appropriate vector. Restriction endonuclease cleavage and electrophoresis experiments indicated that resistant transformants contained plasmids comprising multiple copies of a natural variant allele mediating resistance to copper toxicity. In several cases, the resistance genes were integrated into yeast chromosomal DNA.
As the accelerating pace of development in this area indicates, new selective markers derived from yeast DNA, vectors bearing such markers, and processes for using such vectors are of acute interest as useful tools for genetic manipulation of yeast.